Quality Evaluation of Indoor-Grown Microgreens Cultivated on Three Different Substrates

horticulturae

Article Quality Evaluation of Indoor-Grown Microgreens Cultivated on Three Different Substrates

Roberta Bulgari 1,2,*, Marco Negri 1, Piero Santoro 3 and Antonio Ferrante 1

1 Department of Agricultural and Environmental Sciences, University of Milano, Via Celoria 2, 20133 Milano, Italy; [email protected] (M.N.); [email protected] (A.F.) 2 Department of Agricultural, Forest and Food Sciences, DISAFA, Vegetable Crops and Medicinal and Aromatic Plants, VEGMAP, University of Torino, 10095 Grugliasco, Italy 3 MEG S.r.l., Via Aleardo Aleardi 12, 20154 Milano, Italy; [email protected] * Correspondence: [email protected]

Abstract: The microgreens are innovative products in the horticultural sector. They are appreciated by consumers thanks to their novelty and health-related benefits, having a high antioxidant concentration. This produce can be adopted for indoor production using hydroponic systems. The aim of the present work was to investigate the influence of three growing media (vermiculite, coconut fiber, and jute fabric) on yield and quality parameters of two basil varieties (Green basil—Ocimum basilicum L., Red basil—Ocimum basilicum var. Purpurecsens) and rocket (Eruca sativa Mill.) as microgreens. Microgreens were grown in floating, in a Micro Experimental Growing (MEG®) system equipped with LED lamps, with modulation of both energy and spectra of the light supplied to plants. Results showed high yield, comprised from 2 to 3 kg m−2. Nutritional quality varied among species and higher antioxidant compounds were found in red basil on vermiculite and jute. Coconut fiber allowed the differentiation of crop performance in terms of sucrose and above all nitrate. In particular, Citation: Bulgari, R.; Negri, M.; our results point out that the choice of the substrate significantly affected the yield, the dry matter Santoro, P.; Ferrante, A. Quality Evaluation of Indoor-Grown percentage and the nitrate concentration of microgreens, while the other qualitative parameters were Microgreens Cultivated on Three most influenced by the species. Different Substrates. Horticulturae 2021, 7, 96. https://doi.org/10.3390/ Keywords: coconut fiber; floating system; LED; microgreens; nitrate; plant antioxidants; horticulturae7050096 vermiculite; jute

Academic Editors: Genhua Niu and Celina Gómez 1. Introduction Received: 20 April 2021 In recent years, microgreens have received increasing attention by producers and con- Accepted: 30 April 2021 sumers thanks to their characteristics of tenderness and crunchiness, specific flavors, vivid Published: 2 May 2021 colors, and high nutraceutical value, due to the presence of several bioactive compounds such as antioxidants, vitamins, and macro and micro-minerals [1–8]; for these reasons they Publisher’s Note: MDPI stays neutral are usually considered as functional foods [9,10]. Based on the current literature, there is with regard to jurisdictional claims in also a growing interest from researchers, confirmed by the amount of papers published on published maps and institutional affil- iations. this topic [11]. Microgreens are immature greens, harvested and marketed as soon as the first leaves are developed and the cotyledons are still tender [12]. They can be obtained from vegetables, herbaceous plants, aromatic herbs, and spontaneous species [2,3,13]. Size varies from species to species, but normally they are between 2.5 and 8 cm in height [14]. The growing cycle is short and varies between 7 and 21 days from the emergence of the Copyright: © 2021 by the authors. seedlings. The shoots are harvested by cutting them just above the roots and are eaten raw Licensee MDPI, Basel, Switzerland. either alone or in mixed salads or used as a garnish for dishes. Microgreens can be also This article is an open access article commercialized in boxes with substrates, without harvesting. This strategy allows longer distributed under the terms and conditions of the Creative Commons shelf life and wide opportunity for the commercialization. One of the major limitation Attribution (CC BY) license (https:// of microgreens is their rapid quality deterioration that occurs soon after harvest, and so creativecommons.org/licenses/by/ restricts their commercialization to local sales [6]. From the point of view of cultivation 4.0/). technique, microgreens are very suited for indoor production [8]; they are often grown in

Horticulturae 2021, 7, 96. https://doi.org/10.3390/horticulturae7050096 https://www.mdpi.com/journal/horticulturae Horticulturae 2021, 7, x FOR PEER REVIEW 2 of 14

Horticulturae 2021, 7, 96 cultivation technique, microgreens are very suited for indoor production [8]; they are of-2 of 13 ten grown in hydroponic systems on different substrates [15]. It is important to underline that hydroponic systems could be a sustainable alternative to conventional farming, as they require less water, fertilizer, pesticides, and space for the crops cultivation [16–19]. hydroponic systems on different substrates [15]. It is important to underline that hydro- Moreover,ponic systems in recent could years, be vertical a sustainable farming alternative systems have to conventional emerged as farming, a potential as theysolution require for urbanless water, horticulture, fertilizer, with pesticides, interesting and positive space forimplications the crops in cultivation terms of reduced [16–19]. environ- Moreover, mentalin recent impact years, [20], verticalthanks to farming the shortening systems of have the emergedfood supply as a chain, potential to the solution reduction for urbanof wastehorticulture, and fossil with resources interesting for transportation, positive implications with a in consequent terms of reduced decrease environmental of CO2 [21]. im- Thesepact systems [20], thanks are also to the less shortening affected by of theclimate food change, supply chain,being toperformed the reduction in a protected of waste and environment [22]. fossil resources for transportation, with a consequent decrease of CO2 [21]. These systems areAs also reported less affected in literature, by climate special change, attention being must performed be addressed in a protected to the choice environment of growth [ 22]. media, whichAs reported represents in literature, one of the special key factors attention in the must production be addressed process to the and choice could of influ- growth encemedia, microgreens which representsyield and quality one of [ the23]. key Among factors common in the productionsubstrates used process for the and micro- could in- greensfluence production, microgreens peat-based yield media and quality are the [ 23most]. Among utilized, common followed substratesby coconut used coir and for the severalmicrogreens synthetic production, media. Recently, peat-based natural media fiber are-based the mostmedia utilized,—such followedas jute, cotton, by coconut cellu- coir loseand, etc. several—have syntheticgained increasing media. Recently,popularity natural since they fiber-based could represent media—such a sustainable as jute, cotton,al- ternativecellulose, [3,23 etc.—have]. gained increasing popularity since they could represent a sustainable alternativeThe aim of [3 ,the23]. present work was to investigate the influence of different growing media (vermiculite,The aim of thecoconut present fiber, work and was jute to fabric) investigate on yield the and influence quality of traits different of micro- growing greensmedia of two (vermiculite, basil varieties coconut (Green fiber, basil and— juteOcimum fabric) basilicum on yield andL., Red quality basil traits—Ocimum of microgreens basil- icumof var. two Purpurecsens) basil varieties (Greenand rocket basil— (ErucaOcimum sativa basilicum Mill.). L.,Microgreens Red basil— wereOcimum cultivated basilicum in var. floating,Purpurecsens) with a hydroponic and rocket nutrient (Eruca sativa solution,Mill.). using Microgreens LED illumination, were cultivated in a Micro in floating, Experi- with mentala hydroponic Growing nutrient(MEG® ) solution,platform. using This LEDis anillumination, innovative cultivation in a Micro Experimentalsystem that allows Growing the (MEGmodulation®) platform. of both This energy is an and innovative spectra of cultivation the light supplied system that to plants. allows the modulation of both energy and spectra of the light supplied to plants. 2. Materials and Methods 2. Materials and Methods 2.1. Plant Materials and Sampling 2.1. Plant Materials and Sampling Microgreens of green basil (Ocimum basilicum L.), red basil (Ocimum basilicum ‘Pur- Microgreens of green basil (Ocimum basilicum L.), red basil (Ocimum basilicum ‘Pur- purascens‘), and rocket (Eruca sativa Mill.) were grown at the Faculty of Agricultural and purascens‘), and rocket (Eruca sativa Mill.) were grown at the Faculty of Agricultural and Food Sciences of the University of Milan, in a micro experimental growing chamber Food Sciences of the University of Milan, in a micro experimental growing chamber (MEG®) (MEG® ) (Figure 1). The MEG® is a system equipped with LED-lamps, open-source soft- (Figure1). The MEG ® is a system equipped with LED-lamps, open-source software, auto- ware, automated, developed for home indoor growing. This device utilizes a precision mated, developed for home indoor growing. This device utilizes a precision illumination illumination system, composed of LED diodes managed by a smart control system which system, composed of LED diodes managed by a smart control system which allows the allows the modulation of the light spectrum composition with emission in 454 nm (blue), modulation of the light spectrum composition with emission in 454 nm (blue), 663 (red), 663 (red), and 729 (far-red) and light intensity 65 µmol m−2 s−1, with 12/12 photoperiod and 729 (far-red) and light intensity 65 µmol m−2 s−1, with 12/12 photoperiod (Figure2). (Figure 2). The temperature inside MEG® was 20 °C, and the relative humidity was 60– The temperature inside MEG® was 20 ◦C, and the relative humidity was 60–70%. 70%.

Figure 1. From left to right: Micro Experimental Growing (MEG® ) chamber and harvesting stage Figure 1. From left to right: Micro Experimental Growing (MEG®) chamber and harvesting stage of of indoor-grown microgreens. indoor-grown microgreens.

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FigureFigure 2.2. LLightight spectra spectra utilized utilized for for each each indoor indoor growing growing cycle. cycle. The specific The speciﬁc light lightspectra spectra was meas- was measuredured by using by using a portable a portable spectroradiometer spectroradiometer (Everfine, (Everﬁne, PLA PLA 20). 20).

TheThe three three growing growing substrates substrates used used for for the the experiments experiments were: were: coconut coconut fiber, fiber, vermiculite, vermicu- andlite, jute.and Coconutjute. Coconut fiber fiber is the is mesocarp the mesocarp of Cocos of Cocos nucifera nuciferaL., containing L., containing short, short and medium, and me- lengthdium length fibers leftfibers from left industrial from industrial applications. applications. Depending Depending on origin on and origin industrial and industrial source, theresource, is athere difference is a difference in the physical in the physical and chemical and chemical properties. properties. As reported As reported in literature, in litera- coconutture, coconut fiber possessesfiber possesses remarkable remarkable physical physical and chemical and chemical characteristics—such characteristics— assuch high as water-holdinghigh water-holding capacity, capacity, good drainage,good drainage and aeration, and aeration properties—as properties well—as as well a high as cationa high exchangecation exchange capacity. capacity The pH. The ranged pH range fromd from 5.5 to 5.5 7. to The 7. The porosity porosity was wa 90–95%s 90–95%v/ v/vv.. The The 3 densitydensity waswas 80–100 80–100 kg/m kg/m3 [[2244].]. Vermiculite isis aa silicatesilicate mineral,mineral, thatthat showsshows aa highhigh water water retentionretention andand aa good good aeration. aeration. TheThe pHpH valuevalue was was 7–8, 7–8, the the cation cation exchange exchange capacity capacity was was 3 high.high. The porosity porosity wa wass around around 8 85–95%5–95% v/vv/,v and, and the the de densitynsity wa wass 80– 80–150150 kg/m kg/m3 [24].[ 24Jute]. Jutewas wasa sustainable a sustainable substrate substrate obtained obtained from from organic organic fiber fiber.. Natural Natural fibers fibers have have many many ad- advantages,vantages, such such as as biodegradability biodegradability and and low low costs. costs. A Att present, little technicaltechnical informationinformation 3 waswas available available in in literature literature on on this this substrate. substrate. Jute Jute had had a a density density of of 1300–1500 1300–1500 kg/m kg/m3 [25]. TheThe test test was was carried carried out out in in plastic plastic tanks tanks (35 (3×5 ×27 27× × 1515 cm).cm). AllAll plasticplastic tankstanks containedcontained 2.52.5 L L of of half-strength half-strength Hoagland’s Hoagland’s nutrient nutrient solution. solution. In In each each tank tank was was tested tested a single a single type type of substrate,of substrate, placed placed in aluminum in aluminum trays trays (11 × (119 × ×6.5 9 cm).× 6.5 Eachcm). aluminumEach aluminum tray contained tray contained about 20about g of 20 substrate g of substrate and 2 and g of 2 seeds. g of seeds. The tanksThe tanks were were positioned positioned randomly randomly in the in the chamber. cham- Growingber. Growing cycles cycles ranged ranged from from 12–16 12– days16 days from from sowing, sowing, depending depending on on the the species. species. Two Two growinggrowing cycles cycles were were performed. performed. At At harvest, harvest, yield yield and and dry dry mattermatter percentage percentage (DM%) (DM%) were were calculated,calculated, andand somesome destructivedestructive determinationsdeterminations werewere performedperformed inin laboratory,laboratory, inin order order to evaluate the produce quality. In particular, nitrate, sucrose, chlorophylls, carotenoids, to evaluate the produce quality. In particular, nitrate, sucrose, chlorophylls, carotenoids, anthocyanins, and phenols concentrations were measured. anthocyanins, and phenols concentrations were measured. 2.2. Yield and Dry Matter Percentage 2.2. Yield and Dry Matter Percentage Yield and dry matter percentage were determined by weighing the whole microgreens obtainedYield in and each dry aluminum matter percentage tray (cutting were them determined at the base, by excluding weighing the the substrate), whole micro- be- foregreens and obtained after an in oven-dry each aluminum period (4tray days, (cutting until them reaching at the constant base, excluding weight) the at 75substrate),◦C in a ventilatedbefore and oven. after an oven-dry period (4 days, until reaching constant weight) at 75 °C in a ventilated oven. 2.3. Nitrate 2.3. NitrateNitrates content was measured with the salicylsulphuric acid method [26]. One g fresh sampleNitrates was ground content in 3was mL measured of distilled with water. the The salicylsulphuric extract was centrifuged acid method at 4000 [26 rpm]. One for g 15fresh min sample and the was supernatant ground in was 3 mL recovered of distilled and water. used The for the extrac colorimetrict was centrifuged determination. at 4000 Twentyrpm forµ 15L ofmin sample and the were supernatant added to was 80 µ recoveredL of 5% salicylic and used acid for inthe sulphuric colorimetric acid determi- and to 3nation. mL of Twenty NaOH 1.5µL of N. sample The samples were added were cooledto 80 μL at of room 5% salicylic temperature acid in for sulphuric 15 min andacid theand spectrophotometer to 3 mL of NaOH readings1.5 N. The were samples performed were cooled at 410 nm.at roo Nitratem temperature concentration for 15 was min calculatedand the spectrophotometer referring to a KNO readings3 standard were calibration performed curve at 410 (0, nm. 1, 2.5, Nitrate 5, 7.5, concentration 10 mM KNO was3). calculated referring to a KNO3 standard calibration curve (0, 1, 2.5, 5, 7.5, 10 mM KNO3).

Horticulturae 2021, 7, 96 4 of 13

2.4. Sucrose For the determination of sucrose, 1 g of fresh sample was ground in 3 mL of distilled water. Homogenate was centrifuged at 4000 rpm for 15 min. After that, 0.2 mL of extract was added to 0.2 mL NaOH 2N and incubated at 100 ◦C for 10 min; then 1.5 mL of hot resorcinol solution was added and the sample was incubated at 80 ◦C for 10 min. The resorcinol solution was prepared by adding 35 mg of resorcinol and 90 mg of thiourea in 250 mL HCl 30%, mixed with 25 mL of acetic acid and 10 mL of distilled water. Samples were cooled at room temperature for 15 min and spectrophotometer readings were performed at 500 nm [27]. A calibration curve was built with sucrose standards at 0, 0.5, 1, 1.5, and 2 mM.

2.5. Total Chlorophylls and Carotenoids Chlorophyll a + b and total carotenoids concentrations were determined spectrophotometrically. Frozen shoot tissue (about 1 g) was extracted using 5 mL of 100% (v/v) methanol, for 24 h at 4 ◦C in the dark, followed by quantitative determination of pigments. Absorbance readings were measured at 665.2 and 652.4 nm for chlorophylls and 470 nm for total carotenoids. Pigment concentrations were calculated by Lichtenthaler’s [28] formula and expressed on the basis of tissue fresh weight (FW). Lichtenthaler’s formula using methanol is as follows

chlorophyll a = 16.72 ∗ ABS665.2 − 9.16 ∗ ABS652.4

chlorophyll b = 34.09 ∗ ABS652.4 − 15.28 ∗ ABS665.2 carotenoids = (1000 ∗ ABS470) − (1.63 ∗ [chl a mg/L]) − (104.96 ∗ [chl b mg/L])/221 Chlorophylls and carotenoids concentration calculation

[chl a or chl b or carotenoids]∗volume methanol/mg tissue = µg/mg FW

2.6. Anthocyanins and Phenols For anthocyanins determination, samples of frozen shoot tissue (about 1 g) were ground in pre-chilled mortar and extracted into methanolic HCl (1%). Samples were then incubated overnight at 4 ◦C in the dark. The concentration of cyanidin-3-glucoside equivalents was determined spectrophotometrically at 535 nm [29]. Phenols were spectrophotometrically determined in fresh shoot samples (about 1 g) following the direct measure of the methanolic extract absorbance at 320 nm (phenolic index). Phenolic index was expressed as ABS320 nm g−1 FW.

2.7. Substrate Analysis The dried samples of each substrate were ground into powder with a ZM 100 centrifugal mill equipped with a 0.5 mm mesh sieve (Retsch Gmbh & Co., Haan, Germany) to determine the total nitrogen (N) concentration, the carbon (C) concentration, and C/N ratio, by dry combustion, using a ThermoQuest NA1500 elemental analyser (Carlo Erba, Milan, Italy).

3. Statistical Analysis Statistical analysis was performed using GraphPad Prism version 6 for Windows (GraphPad Software; La Jolla, CA, USA, www.graphpad.com (accessed on 25 April 2021). Data are the result of the average of two different growing cycles, which took place under the same experimental conditions. The reported values are means with standard errors (SE) of n = 6 biological replicates. All data were subjected to two-way ANOVA and differences among means were determined by Tukey’s multiple comparison test (p < 0.05).

4. Results 4.1. Yield and Dry Matter Percentage The ANOVA analysis revealed that the interaction between the two factors (substrate x species) and the two factors (substrate and species) were statistically signiﬁcant Horticulturae 2021, 7, 96 5 of 13

(Table1 ). The highest yield was observed in rocket microgreens grown on jute substrate (3201.09 g/m2). In general, red basil showed the lowest yields (with a minimum of 2008.38 g/m2 on jute), while green basil showed intermediate values (Figure3). Based on average data, rocket species showed higher yields (around 3000 g/m2) compared to the two basil varieties tested. The species with the lowest yields was red basil (average yield around 2200 g/m2). Also, in the case of the dry matter percentage calculation, the two- way ANOVA highlighted signiﬁcant differences both for interaction and factors (Table1) . Values ranged from 3.8% (for rocket grown on jute) to 6.6% (in the case of red basil on coconut ﬁber). Rocket and red basil grown on jute showed the lowest values of dry matter percentage (Figure4).

Table 1. Summary of the results of the two-way ANOVA for all the analyzes performed. Data were subjected to two-way ANOVA and Tukey’s multiple comparison test was used for evaluating the differences among means at (* p < 0.05, *** p < 0.001, **** p < 0.0001). NS = not signiﬁcant.

Determination Source of Variation p Value p Value Summary Interaction (SubxSp) <0.0001 **** Yield Substrate <0.0001 **** Species <0.0001 **** Interaction (SubxSp) <0.0001 **** Dry matter Substrate <0.0001 **** Species <0.0001 **** Interaction (SubxSp) 0.759 NS Nitrate Substrate 0.001 ** Species 0.292 NS Interaction (SubxSp) 0.183 NS Sucrose Substrate 0.913 NS Species 0.032 * Interaction (SubxSp) 0.122 NS Chlorophyll a + b Substrate 0.216 NS Species 0.112 NS Interaction (SubxSp) 0.483 NS Carotenoids Substrate 0.120 NS Species 0.019 * Interaction (SubxSp) 0.042 * Anthocyanins Substrate 0.266 NS Species 0.0001 *** Horticulturae 2021, 7, x FOR PEER REVIEW Interaction (SubxSp) 0.136 NS 6 of 14 Phenolic index Substrate 0.397 NS Species 0.018 *

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Figure 3. Yield of green basil, rocket, and red basil microgreens grown on three different substrates Figure 3. Yield of green basil, rocket, and red basil microgreens grown on three different sub- (coconut ﬁber, vermiculite, and jute). Values, subjected to two-way ANOVA, are means ± SE (n = 6). strates (coconut fiber, vermiculite, and jute). Values, subjected to two-way ANOVA, are means ± SE (n = 6).

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Figure 4. Dry matter of green basil, rocket, and red basil microgreens grown on three different substrates. Values, subjected to two-way ANOVA, are means ± SE (n = 6).

4.2. Nitrate Concentration The two-way ANOVA analysis showed that the interaction and the species factor were not significant; on the contrary, the differences among substrates were significant (Table 1). Nitrate concentration was lower in microgreens grown on coconut fiber compared to other substrates; in particular, the lowest value was observed in red basil microgreens (687.37 mg/kg FW). The three species cultivated on vermiculite and jute showed similar levels, ranging between 1191.12 and 1363.13 mg/kg FW (Figure 5).

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Figure 3. Yield of green basil, rocket, and red basil microgreens grown on three different sub- Horticulturae 2021, 7, 96 strates (coconut fiber, vermiculite, and jute). Values, subjected to two-way ANOVA, are means6 ± of 13 SE (n = 6).

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Figure 4. Dry matter of green basil, rocket, and red basil microgreens grown on three different Figure 4. Dry matter of green basil, rocket, and red basil microgreens grown on three different substrates. Values, subjected to two-way ANOVA, are means ± SE (n = 6). substrates. Values, subjected to two-way ANOVA, are means ± SE (n = 6). 4.2. Nitrate Concentration 4.2. NitrateThe two-wayConcentration ANOVA analysis showed that the interaction and the species factor were notThe significant; two-way on ANOVA the contrary, analysis the differences showed that among the substratesinteraction were and significantthe species(Table factor1) . wereNitrate not concentrationsignificant; on wasthe contrary, lower in the microgreens differences grown among on substrates coconut fiber were compared significant to Horticulturae 2021, 7, x FOR PEER REVIEW(Tableother 1 substrates;). Nitrate concentration in particular, was the lowestlower in value microgreens was observed grown in on red coconut basilmicrogreens fiber com-7 of 14 pared(687.37 to other mg/kg substrates; FW). The in three particular, species the cultivated lowest value on vermiculite was observed and jutein red showed basil micro- similar greenslevels, (687.37 ranging mg/kg between FW). 1191.12 The three and 1363.13species mg/kgcultivated FW on (Figure vermiculite5). and jute showed similar levels, ranging between 1191.12 and 1363.13 mg/kg FW (Figure 5). G re e n b a s il 2 0 0 0 R o c k e t

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Figure 5. Nitrate concentration of green basil, rocket, and red basil microgreens grown on three Figure 5. Nitrate concentration of green basil, rocket, and red basil microgreens grown on three different substrates. Values, subjected to two-way ANOVA, are means ± SE (n = 6). different substrates. Values, subjected to two-way ANOVA, are means ± SE (n = 6). 4.3. Sucrose Levels 4.3. Sucrose Levels The analysis of sucrose concentration allowed highlighting that the species factor wasThe significant analysis (Tableof sucrose1). Values concentration ranged fromallowed 320 highlighting to around 500 that mg/kg the species FW. Green factor basilwas significantmicrogreens (Table contained 1). Values the ranged lowest from levels. 320 Theto around highest 500 concentration mg/kg FW. Green was foundbasil micro- in red greensbasil microgreenscontained the grown lowest on levels. coconut The fiber highest (484.35 concentration mg/kg FW). was The found coconut in red fiber basil induced mi- crogreensdifferent grown crop performance. on coconut fiber Rocket (484.35 microgreens mg/kg FW). showed The thecoconut same fiber sucrose induced concentrations, different croparound performance. 400 mg/kg Rocket FW (Figure microgreens6). showed the same sucrose concentrations, around 400 mg/kg FW (Figure 6).

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Figure 6. Sucrose concentration of green basil, rocket, and red basil microgreens grown on three different substrates. Values, subjected to two-way ANOVA, are means ± SE (n = 6).

4.4. Total Chlorophylls and Carotenoids With regard to chlorophylls a + b concentration, the two-way ANOVA analysis showed that interaction and factors were not statistically significant (Table 1), with values ranging from 202.51 to 316.42 µg/g FW (Figure 7). In general, lower chlorophyll concentrations were observed in red basil, without difference among the substrates. The higher values were found in green basil microgreens (Figure 8), although the highest concentration was obtained for rocket grown on coconut fiber. Regarding carotenoids, it is possible to observe a significant effect deriving from the species (Table 1); the lowest concentrations were measured in red basil microgreens, while the other two species showed similar values (Figure 9).

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Figure 5. Nitrate concentration of green basil, rocket, and red basil microgreens grown on three different substrates. Values, subjected to two-way ANOVA, are means ± SE (n = 6).

4.3. Sucrose Levels The analysis of sucrose concentration allowed highlighting that the species factor was significant (Table 1). Values ranged from 320 to around 500 mg/kg FW. Green basil microgreens contained the lowest levels. The highest concentration was found in red basil microgreens grown on coconut fiber (484.35 mg/kg FW). The coconut fiber induced different crop performance. Rocket microgreens showed the same sucrose concentrations, around Horticulturae 2021, 7, 96 400 mg/kg FW (Figure 6). 7 of 13

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Figure 6. Sucrose concentration of green basil, rocket, and red basil microgreens grown on three Figure 6. Sucrose concentration of green basil, rocket, and red basil microgreens grown on three different substrates. Values, subjected to two-way ANOVA, are means ± SE (n = 6). different substrates. Values, subjected to two-way ANOVA, are means ± SE (n = 6). 4.4. Total Chlorophylls and Carotenoids 4.4. Total Chlorophylls and Carotenoids With regard to chlorophylls a + b concentration, the two-way ANOVA analysis showed thatWith interaction regard and to factorschlorophylls were nota + statistically b concentration, significant the (Table two-way1), with ANOVA values analysis ranging showedfrom 202.51 that interaction to 316.42 µg/g and FW factors (Figure were7). not In general,statistically lower significant chlorophyll (Table concentrations 1), with values were rangingobserved from in red 202.51 basil, to without 316.42 differenceµg/g FW among(Figure the7). substrates.In general, The lower higher chlorophyll values were concen- found trationsin green were basil observed microgreens in red (Figure basil,8 ),without although difference the highest among concentration the substrates. was obtainedThe higher for values were found in green basil microgreens (Figure 8), although the highest concentra- Horticulturae 2021, 7, x FORHorticulturae PEER REVIEW 2021, 7 , x FOR PEER REVIEWrocket grown on coconut fiber. Regarding carotenoids, it is possible9 toof observe15 a significant8 of 14 tioneffect was deriving obtained from for therocket species grown (Table on coco1); thenut lowest fiber. concentrationsRegarding carotenoids, were measured it is possible in red tobasil observe microgreens, a significant while effect the other deriving two species from the showed species similar (Table values 1); the (Figure lowest9). concentrations were measured in red basil microgreens, while the other two species showed similar

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Figure 8. From left toFigure right: green8. Fro mbasil left microgreens to right: green grown basil on microgreens jute, vermiculite grown and on jute,coconut vermiculite fiber. and coconut fiber. Figure 8. From left to right: green basil microgreens grown on jute, vermiculite and coconut ﬁber. This species showed Thishigh speciesconcentrations showed ofhigh chlorophyll concentration a + b.s of chlorophyll a + b. This species showed high concentrations of chlorophyll a + b.

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Figure 9. Carotenoid Figureconcentrations 9. Carotenoid of green concentration basil, rocket,s of and green red basil, basil rocket,microgreens and red grown basil on microgreens grown on three different substrates.three Values,different subjected substrates. to two-wayValues, subjected ANOVA, to are two means-way ±ANOVA, SE (n = 6). are means ± SE (n = 6).

4.5. Anthocyanins and4.5. Phenolic Anthocyanins Index and Phenolic Index Statistical analysis Statisticalshowed that analysis for anth showedocyanins that the for interactionanthocyanins between the interaction substrate between substrate and species was significantand species for wasp < 0.05 significant (Table 1). for The p < species0.05 (Table factor 1). had The aspecies significant factor effect had a significant effect for p < 0.0001. As couldfor p

Horticulturae 2021, 7, x FOR PEER REVIEW 9 of 15

Figure 7. Chlorophyll a + b concentration of green basil, rocket, and red basil microgreens grown on three different substrates. Values, subjected to two-way ANOVA, are means ± SE (n = 6).

Figure 8. From left to right: green basil microgreens grown on jute, vermiculite and coconut fiber. Horticulturae 2021, 7, 96 8 of 13 This species showed high concentrations of chlorophyll a + b.

Figure 9. Carotenoid concentrations of green basil, rocket, and red basil microgreens grown on three Figure 9. Carotenoid concentrations of green basil, rocket, and red basil microgreens grown on three different substrates.different Values, substrates. subjected Values, to two-way subjected ANOVA, to two-way are means ANOVA, ± SE (n are = means6). ± SE (n = 6). 4.5. Anthocyanins and Phenolic Index 4.5. Anthocyanins and Phenolic Index Statistical analysis showed that for anthocyanins the interaction between substrate and Statistical analysis showed that for anthocyanins the interaction between substrate species was significant for p < 0.05 (Table1). The species factor had a significant effect for and species was significant for p < 0.05 (Table 1). The species factor had a significant effect Horticulturae 2021, 7, x FOR PEER REVIEWp <0.0001. As could be expected, red basil microgreens showed higher anthocyanins9 of levels 14 (up Horticulturae 2021for, 7, x p FOR < 0.0001. PEER REVIEW As could be expected, red basil microgreens showed higher anthocyanins 9 of 14 to 27.16 mg/100 g FW) compared to rocket and green basil, in particular this species grown on levels (up to 27.16 mg/100 g FW) compared to rocket and green basil, in particular this vermiculite and jute showed the highest values. The lowest concentration can be found in green species grown on vermiculite and jute showed the highest values. The lowest concentration can be foundonbasil in vermiculite green on jute basil (9.89 (Figure on mg/100jutes 11(9. 89an g dmg/100 FW) 12) and (Figure g juteFW) 10reached (Figure). The speciesthe10). highest The factor species levels was factor (6.44 significant and 7.37 also ABS in320 the case on vermiculite (Figures 11 and 12) and jute reached the highest levels (6.44 and 7.37 ABS320 was significant nmalsoof/gphenolic respectively);in the case index of greenphenolic (Table basil1 ).index Redshowed (Table basil in microgreens general1). Red basillower grown microgreens values on compared vermiculite grown to the(Figures other spe-11 and 12) nm/g respectively); green basil showed in general lower values compared to the other spe- ciesand considered jute reached. the highest levels (6.44 and 7.37 ABS320 nm/g respectively); green basil showed ciesin generalconsidered lower. values compared to the other species considered.

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h 0 r e e e it t P b l u i r u e J f e t e ic it t b l u u i m u J n f r c o t e i c u V m o n r C o e c V o C Figure 11. Phenolic index values of green basil, rocket, and red basil microgreens grown on three Figure 11. Phenolic index values of green basil, rocket, and red basil microgreens grown on three Fdifferentiguredifferent 11. substrates. Phenolic substrates. index Values, Values, values subjected subjected of green to two basil, to two-way-way rocket, ANOVA, ANOVA,and red are basil means are microgreens means ± SE (n± =SE 6). grown (n = 6). on three different substrates. Values, subjected to two-way ANOVA, are means ± SE (n = 6).

Figure 12. Red basil microgreens grown on vermiculite substrate. This species reached the highest Flevelsigure of 12. phenolic Red basil compounds. microgreens grown on vermiculite substrate. This species reached the highest levels of phenolic compounds.

Horticulturae 2021, 7, x FOR PEER REVIEW 9 of 14

on vermiculite (Figures 11 and 12) and jute reached the highest levels (6.44 and 7.37 ABS320 nm/g respectively); green basil showed in general lower values compared to the other species considered.

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Figure 12. Red basil microgreens grown on vermiculite substrate. This species reached the highest Figure 12. Red basil microgreens grown on vermiculite substrate. This species reached the highest levels of phenolic compounds. levels of phenolic compounds.

4.6. Substrate Analysis The total N was higher in coconut ﬁber, with 0.49%, followed by vermiculite, with 0.31%, while no nitrogen was detected in jute (Table2). The C concentration was higher in vermiculite and coconut ﬁber, with 44.25 and 41.31% respectively, while 0.28% was found in jute. The highest C/N ratio was observed in vermiculite substrate, followed by jute (Table2).

Table 2. Total N and C percentage, and C/N ratio in the different substrates utilized for the trial. Values are means (n = 2).

Substrate N (%) C (%) C/N Coconut ﬁber 0.49 41.31 85.10 Vermiculite 0.31 44.25 144.15 Jute 0 0.28 /

5. Discussion The growing medium plays a very important role in determining the microgreens’ yield and quality [23], and the sustainability of the production process. Associated with this, in the last few years, an increased number of scientific papers reported the beneficial effects of LED light, in controlled environment agriculture, on plant growth and quality traits, including the accumulation of molecules of interest, such as carotenoids, phenolics, and glucosinolates [7,8,30–33]. In the present work, different growing media usable for indoor microgreens cultivation were evaluated: vermiculite (inorganic material), coconut fiber (a natural, organic fiber), and jute (organic by-product and discarded material from several industrial processes). At present, peat-based substrates are the main growing media used for microgreens cultivation, but they are expensive and non-renewable. An alternative to peat may be coconut coir, an organic and renewable resource. However, coir has some disadvantages, in terms of possible high concentration of salts, as well as high fungal and bacterial counts [23,34]. A low-cost and renewable alternative could be the use of fibrous materials—such as polyester, cotton, or jute fiber. Other available inorganic media are for example perlite, vermiculite, and rockwool but these media are expensive, their production is energy demanding, and they are not easily disposable at the end of the production. In our experiment, the analysis of the performance of the three different substrates has also the aim of identifying alternatives that could be more environmentally friendly and cost effective, as by-products from industrial processes. Certainly, the fresh yield is a key factor in the cultivation of microgreens, if we consider that they are typically sold on a “per-FW” basis [35]. A low yield continues to be a limiting factor for microgreen industry [1]. Data related to fresh yield were similar or Horticulturae 2021, 7, 96 10 of 13

slightly higher than that reported in literature for microgreens [1,8,13,35–37]. Specifically, Bulgari et al. [1] obtained a yield of 1 kg/m2 for basil microgreens and around 1.5 kg/m2 for rocket, and Kyriacou et al. [38] produced 1.6 kg/m2 of green basil microgreens and 3 kg/m2 of red basil. In our trial, good results were especially obtained for rocket, on all the three substrates tested, with the highest data observed on jute. The dry matter percentage showed an opposite behavior; in fact, rocket microgreens had the lowest DM% in all the three substrates tested. A significant effect, resulting from species and substrates and their interaction, was therefore noticed in our experiment on these quantitative parameters, and the results underline the importance of growth media. Regarding qualitative parameters, nitrates are among the main compounds that determine foods healthiness. Vegetables can accumulate different concentrations of nitrate – (NO3 ) and this compound could consequently cause health problems in human [39]. – Microgreens can be considered a good source of minerals and their NO3 concentration is generally very low. Several studies reported that the nitrate in microgreens had lower levels than in mature salads [8,13,40,41]. Thus, microgreens can be safely consumed for a healthy diet, even in the case of children, avoiding harmful phenomena such as methemoglobinemia. Our findings showed that nitrate concentrations ranged from 687.37 to 1363.13 mg/kg FW; the substrate factor had a significant effect on the concentration. A marked reduction of nitrate was in particular observed on coconut fiber. Microgreens are confirmed as non-nitrate accumulators [11]. As reported by Di Gioia et al. [23], the – choice of the substrate can limit microgreens NO3 accumulation thus influencing the produce quality. Regarding green and red basil, we have found lower levels, and in some cases halved, compared to the data reported in the experiment of Kyriacou et al. [38], who studied 13 microgreens species grown in a growth chamber, on a commercial peat-based substrate. They observed that nitrate levels can vary considerably across species; however, the species factor did not significantly affect our results. The C/N is usually considered a parameter that can affect the nitrate leaf accumulation. However, the obtained results cannot be justified by the C/N, due to the limited growing period. The light type could also induce different responses in plants and influence the nitrate content [8]; with regards to this, an accurate choice of the substrate combined with LED illumination can be a good strategy to get produce rich in molecules of interest and with lower amounts of nitrate. The sucrose concentration allowed highlighting that the species factor was relevant; the highest concentration was found in red basil grown on coconut fiber. Sucrose is important for microgreens preservation during the distribution chain and storage. In the case of microgreens, differences in the sugars level can be attributed to variations in the genotype [11]. Furthermore, different lighting conditions during the growing cycle can also affect the content of carbohydrates, resulting from a stimulation of the photosynthetic process [30]. It is interesting to note that sucrose levels are moderate and lower compared to baby leaf and adult vegetable leaves [42]. Such low sugars concentration can explain the very short shelf life (1–2 days) of microgreens [43,44]. However, we found higher values than those described by Bulgari et al. [1] related to microgreens cultivated outdoor, on vermiculite; this denotes a possible effect derived by the light quality. As previously reported by Lin et al. [45], a specific LED light can be used to enhance the sucrose concentration and the nutritional profile of vegetables. Moving to the chlorophylls, the content of these pigments in vegetables is moreover important for the visual appearance of the produce. Color and appearance determine if a product is accepted or rejected by the consumer, and these aspects are even more relevant in a product like microgreens, highly appreciated also for their colors [46]. Chlorophylls play a significant role in photosynthesis, since they represent part of light-harvesting complex. As reported in literature, significant genotypic variations were observed for chlorophylls, and the levels can be also influenced by the light conditions [11]. Considering that microgreens are mainly composed by cotyledons, it is evident that lower concentrations of chlorophylls, carotenoids, phenols, and anthocyanins were detected as opposed to baby leaf or adult vegetables of the same species [47,48]. Red basil microgreens reached, in general, the Horticulturae 2021, 7, 96 11 of 13

highest levels of phenolic compounds compared to the other species. Purple varieties are characterized by the accumulation of anthocyanins in leaves and ﬂowers, mostly at adult stage [49]. Total phenolics are higher in the purple basils than in the green cultivars [50] and this was conﬁrmed also at microgreens stage, as shown by our data. Purple basil is a very good natural source of anthocyanins, which are correlated with prevention of diverse human diseases. The obtained results for red basil microgreens grown on vermiculite and jute look promising to get a product with a high nutraceutical value.

6. Conclusions As we have witnessed over the past few years, the interest in microgreens has increased [11]. They are appreciated by consumers thanks to their novelty and health-related benefits, being rich in antioxidant compounds. Moreover, microgreens can be easily grown by people for home use, or be produced at a larger scale, with indoor grow systems. In- door plant cultivation systems are emerging because they allow to produce fresh food in urban environments and unfavorable climatic contexts [18,51]. The sustainability of the cultivation system is a critical point of indoor production, which can be improved by the optimization of the production factors, such as water, substrate, and energy in- take [51]. In particular, energy consumption mainly depends on the lighting, since the optimization of light emission—in terms of quality and intensity—can improve the system sustainability [52,53]. The choice of the substrate is no less important, as we have seen. In addition to the economic and environmental impact, the substrate can influence the produce quality [23]. According to this, we included different substrates in the present work, with different characteristics, in order to suggest valid alternatives in the choice of the growing media, maintaining at the same time a good final quality of the product. Our results point out that the choice of the substrate significantly affected the yield, the dry matter percentage and the nitrate concentration of microgreens. On the contrary, the other qualitative parameters were most influenced by the species. Certainly, additional studies may be needed to evaluate the effect of these substrates (vermiculite, coconut fiber, and jute fabric) on the production and quality traits (nutritional value, color, texture, taste, etc.) of microgreens, also in relation to the determination of the optimal protocol for LED management, to identify the best cultivation conditions. Moreover, this study has provided interesting preliminary data on the use of red basil for the production of microgreens, a species that has been little studied to date for this type of production.

Author Contributions: R.B., substantial contribution to the experimental work, interpretation of data, and drafting of the manuscript; M.N., participated in the experimental work, drafting, and revision of the manuscript; P.S., experimental setup of the Micro Experimental Growing (MEG®) platform for microgreens cultivation; A.F., experimental design and coordination of the work, interpretation of data, drafting and revision of the manuscript. All authors read and approved the final version of the manuscript. Funding: This work was funded by the INNODRIVER—Lombardy Region funds project entitled “Ot- timizzazione di Micro Experimental Growing (MEG®) per la produzione di Microgreens-OPTIMEG”. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Data reported in the manuscript can be provided upon request. Conflicts of Interest: The authors declare no conflict of interest.

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